Throughout the application, various publications are referenced in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in the application in order to more fully describe the state of the art to which this invention pertains.
1. Field of the Invention
The present invention is related to the biomedical arts, in particular to genetics.
2. Discussion of the Related Art
The particular gene that is the subject of the present invention is PTTG, which is believed to have a role in proper cell cycle progression.
Events such as DNA synthesis, chromosome segregation, spindle assembly, cytokinesis, and other aspects of cell division, must be executed in ordered sequence during the cell cycle. Proper cell cycle progression is a complex process requiring many cell cycle regulators including p53, Rb, cyclins, cdks and cdk inhibitors p21, and p16, among others. (Reddel, R. R., Genes involved in the control of cellular proliferative potential, Ann. N.Y. Acad. Sci. 854: 8-19 [1998]; Prosperi, E., Multiple roles of the proliferating cell nuclear antigen: DNA replication, repair and cell cycle control, Prog. Cell Cycle Res. 3:193-210 [1997]). Loss or mutation of these genes leads to dysfunctions of cell cycle progression and are frequently involved in tumorigenesis and apoptosis resulting in pathological consequences. For example, mice lacking p53 show unregulated G1 checkpoint control and a high prevalence of spontaneous tumor development (Donehower, L. A., et al., Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors, Nature 356:215-221 [1992]); mice lacking Rb do not survive fetal development while Rb+/− mice developed pituitary tumors at 8 months (Jacks, T., et al., Effects of an Rb mutation in the mouse, Nature 359:295-300 [1992]); mice lacking p21 undergo normal development but show defective G1 checkpoint control (Deng, C., et al., Mice lacking p21CIP1WAFI undergo normal development, but are defective in G1 checkpoint control, Cell 82:675-684 [1995]).
Recently, a family of proteins including securins, separins and cohesins were found to play important roles during sister chromatid separation in eukaryotic cell cycle M phase (Nasmyth, K., et al., Splitting the chromosome: cutting the ties that bind sister chromatids, Science 288:1379-1384 [2000]). These proteins exhibit characteristics of cell cycle regulators. Securins (e.g., S. cerevisine Pds1p, S. prombe Cut2, Drosophila PIM, Xenopus securin) share at least one destruction box and a nine amino acid consensus motif [RX(A or V or L) LGXXX N], originally identified in B-type cyclins. (Ciosk, R., et al., An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast, Cell 93:1067-1076 [1998]; Funabiki, H., et al., Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes, Embo. J. 15:6617-6628 [1996]; Zou, H., et al., Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis, Science 285:418-422 [1999]; Stratmann, R., et al., Separation of sister chromatids in mitosis requires the Drosophila pimples product, a protein degraded after the metaphase/anaphase transition, Cell 84: 25-35 [1996]). The separins (Esp1p, Cut1, BimB) share a conserved carboxy-terminal domain, which binds to securins (Ciosk, R., et al., An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast, Cell 93:1067-1076 [1998]; Funabiki, H., et al., Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes, Embo. J. 15:6617-6628 [1996]; May, G. S., et al., The bimB3 mutation of Aspergillus nidulans uncouples DNA replication from the completion of mitosis, J. Biol. Chem. 267:15737-15743 [1992]).
Securins reach their highest expression level in M phase. Securins accumulate during interphase, and they bind to separin, which prevents premature separin activation. In a normal cell cycle, anaphase promoting complex (APC) eventually degrades securins, thus activating separin to facilitate chromosome segregation. (Nasmyth, K., et al., Splitting the chromosome: cutting the ties that bind sister chromatids, Science 288:1379-1384 [2000]). In this sense, securins function as inhibitors of chromatid separation during anaphase.
To date, characterization of mammalian securin or separin has been limited. (Zou, H., et al., Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis, Science 285:418-422 [1999]).
However, pituitary tumor transforming gene (PTTG) (Pei, L., et al., Isolation and characterization of a pituitary tumor-transforming gene (PTTG), Mol. Endo. 11:433-441 [1997]), a recently described oncogene isolated from pituitary tumor growth hormone-secreting cells by differential display, has 44.6% amino acid identity with Xenopus securin. Indeed, PTTG has one destruction box (RXLGXXXN) and cyclin B-like 9 amino acid consensus motif. PTTG protein preferentially localizes in the cell nucleus. (Yu, R., et al., Pituitary tumor transforming gene (PTTG) regulates placental JEG-3 cell division and survival: evidence from live cell imaging, Mol. Endo. 14:1137-1146 [2000]). Expression levels of PTTG protein change in a temporal pattern during cell cycle progression, peaking during M phase; PTTG is phosphorylated by cdc2 and MAPK. (Ramos-Morales, F., et al., Cell cycle regulated expression and phosphorylation of hpttg proto-oncogene product, Oncogene 19:403-409 [2000]; Pei, L., Activation of mitogen-activated protein kinase cascade regulates pituitary tumor transforming gene transactivation function, J. Biol. Chem. 275:31191-31198 [2000]). PTTGs have been identified in rat, mouse, and human cells. (e.g., PCT/US97/21463; Wang, Z., et al., Pituitary tumor transforming gene (PTTG) transforming and transactivation activity, J. Biol. Chem. 275:7459-7461 [2000]).
PTTG1, the PTTG equivalent in humans, is expressed at low levels in most normal human tissues. (Chen, L. et al., Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization, Gene. 248:41-50 [2000]; Heaney, A. P. et al. [2000]). PTTG is abundant only in normal testis and thymus. (Wang, Z., et al., Characterization of the murine pituitary tumor transforming gene (PTTG) and its promoter, Endocrinology 141:763-771 [2000]). When expressed at normal levels, PTTG mediates promoter transcriptional activation. (Wang, Z., et al., Pituitary tumor transforming gene (PTTG) transforming and transactivation activity, J. Biol. Chem. 275:7459-7461 [2000]), utilizes c-myc as its downstream gene target. By dysregulating chromatid separation, PTTG overexpression also leads to aneuploidy, i.e., cells having one or a few chromosomes above or below the normal chromosome number (Zou et al. [1999]; Yu, R. et al. [2000]). At the end of metaphase, securin is degraded by an anaphase-promoting complex, releasing tonic inhibition of separin, which in turn mediates degradation of cohesins, the proteins that hold sister chromatids together. Overexpression of a nondegradable PTTG disrupts sister chromatid separation (Zou et al. [1999]) and overexpression of PTTG causes apoptosis and inhibits mitoses (Yu, R. et al. [2000]). The securin function of PTTG suggests that PTTG may also be expressed in normal proliferating cells. In adult humans, PTTG1 mRNA is most abundant in testis, an organ containing rapidly proliferating gametes. (Zhang, X. et al. [1999a]); Wang, Z. et al. [2000]).
In contrast, PTTG1 is highly expressed in human tumors and is responsive to estrogen induction. (Zhang, X., et al., Structure, expression, and function of human pituitary tumor-transforming gene (PTTG), Mol. Endo. 13:156-166 [1999]; Heaney, A. P., et al., Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis, Nature Med. 5:1317-1321 [1999]). Indeed, PTTG is highly expressed in pituitary tumors and neoplasms from the hematopoietic system and colon, and PTTG is considered to be a proto-oncogene, because PTTG overexpression in NIH3T3 cells induces cell transformation and in vivo tumor formation. (Pei, L., et al., Isolation and characterization of a pituitary tumor-transforming gene (PTTG), Mol. Endo. 11:433-441 [1997]; Zhang, X. et al., Structure, expression, and function of human pituitary tumor-transforming gene (PTTG), Mol. Endocrinol. 13:156-66 [1999a]; Zhang, X. et al., Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas, J. Clin. Endocrinol. Metab. 84:761-67 [1999b]; Heaney, A. P. et al., Pituitary tumor transforming gene in colorectal tumors, Lancet 355:712-15 [2000]; Dominguez, A. et al., hPTTG, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG, Oncogene 17:2187-93 [1998]; Saez, C. et al., hPTTG is over-expressed in pituitary adenomas and other primary epithelial neoplasias, Oncogene 18:5473-6 [1999]). In addition, PTTG has been shown to possess other physiological roles in mammals, although mechanisms are unclear.
PTTG also has been shown to upregulate basic fibroblast growth factor secretion (Zhang, X. et al. [1999a]), and transactivate DNA transcription (Dominguez, A. et al. [1998]; Wang, Z. et al., Pituitary tumor transforming gene (PTTG) transactivating and transforming activity, J. Biol. Chem. 275:7459-61[2000]).
The recent discovery of human PTTG2 gene, which shares high sequence homology with human PTTG1, implies the existence of a PTTG family. (Prezant, T. R., et al., An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family, J. Clin. Endocrinol. Metab. 84:1149-1152 [1999]). There is evidence that a PTTG family consists of at least three genes that share a high degree of sequence homology, including human PTTG1, located on chromosome 5q33. (Id.). Murine PTTG shares 66% nucleotide base sequence homology with human PTTG1 and also exhibits transforming ability. (Wang, Z. and Melmed, S., Characterization of the murine pituitary tumor transforming gene (PTTG) and its promoter, Endocrinology 14:763-771 [2000].
Despite all of the research and resources applied to understanding the role of PTTG in cell cycle control and the pathogenesis of numerous disease conditions, including tumorigenesis, the function and mode of action of PTTG in vivo remains poorly understood. This is due, in part, to the fact that there has been no readily available and effective in vivo model for studying PTTG.
In a recently published article, researchers claim to have obtained three “securin-null” mice of both sexes. (Mei, J., Huang, X., and Zhang, P., Securin is not required for cellular viability, but is required for normal growth of mouse embryonic fibroblasts, Current Biology 11:1197-1201 [2001]). The securin referred to is represented to be PTTG. Aside from the observation that PTTG −/− MEFs exhibited delayed cell cycle progression of G2-M phase, no other phenotypic differences from wild-type were observed in either the PTTG −/− mice or the cells derived therefrom.
There remains a need for an in vivo model for studying the role of PTTG in mammalian physiology at the cellular, tissue, and/or organismal level, including the study of diabetes, cell cycle control, oncogenesis, and various other medical conditions and phenomena relating to PTTG expression. This and other benefits are provided by the present invention as described herein.